Aged rhesus monkeys exhibit deficits in hippocampus-dependent memory, similar to aging humans. Here we explored the basis of cognitive decline by first testing young adult and aged monkeys on a standard recognition memory test (delayed nonmatching-to-sample test; DNMS). Next we quantified synaptic density and morphology in the hippocampal dentate gyrus (DG) outer (OML) and inner molecular layer (IML). Consistent with previous findings, aged monkeys were slow to learn DNMS initially, and they performed significantly worse than young subjects when challenged with longer retention intervals. Although OML and IML synaptic parameters failed to differ across the young and aged groups, the density of perforated synapses in the OML was coupled with recognition memory accuracy. Independent of chronological age, monkeys classified on the basis of menses data as peri/post-menopausal scored worse on DNMS, and displayed lower OML perforated synapse density, than pre-menopausal monkeys. These results suggest that naturally occurring reproductive senescence potently influences synaptic connectivity in the DG OML, contributing to individual differences in the course of normal cognitive aging.
delayed nonmatching-to-sample; disector method; estrogen; hippocampus; menopause; outer molecular layer; perforated synapse; post-synaptic density; recognition memory
Rhesus monkeys provide a valuable model for studying the neurobiological basis of cognitive aging, because they are vulnerable to age-related memory decline in a manner similar to humans. In this study, young and aged monkeys were first tested on a well-characterized recognition memory test (delayed nonmatching-to-sample; DNMS). Then, electron microscopic immunocytochemistry was performed to determine the subcellular localization of two proteins in the hippocampal dentate gyrus (DG): the GluA2 subunit of the glutamate alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptor and the atypical protein kinase C ζ isoform (PKMζ). PKMζ promotes memory storage by regulating GluA2-containing AMPA receptor trafficking. Thus, we examined whether the distribution of GluA2 and PKMζ is altered with aging in DG axospinous synapses and whether it is coupled with memory deficits. Monkeys with faster DNMS task acquisition and more accurate recognition memory exhibited higher proportions of dendritic spines coexpressing GluA2 and PKMζ. These double-labeled spines had larger synapses, as measured by postsynaptic density area, than single- and unlabeled spines. Within this population of double-labeled spines, aged monkeys compared to young expressed a lower density of synaptic GluA2 immunogold labeling, which correlated with lower recognition accuracy. Additionally, higher density of synaptic PKMζ labeling in double-labeled spines correlated with both faster task acquisition and better retention. Together, these findings suggest that age-related impairment in maintenance of GluA2 at the synapse in the primate hippocampus is coupled with memory deficits.
AMPA receptor; delayed nonmatching-to-sample test; GluR2; immunogold; PKMζ; recognition memory
In the past few decades it has become clear that estrogen signaling plays a much larger role in modulating the cognitive centers of the brain than previously thought possible. We have developed a nonhuman primate (NHP) model to investigate the relationships between estradiol (E) and cognitive aging. Our studies of cyclical E treatment in ovariectomized (OVX) young and aged rhesus monkeys have revealed compelling cognitive and synaptic effects of E in the context of aging. Delayed response (DR), a task that is particularly dependent on integrity of dorsolateral prefrontal cortex (dlPFC) area 46 revealed the following: 1) that young OVX rhesus monkeys perform equally well whether treated with E or vehicle (V), and 2) that aged OVX animals given E perform as well as young adults with or without E, whereas OVX V-treated aged animals display significant DR impairment. We have analyzed the structure of layer III pyramidal cells in area 46 in these same monkeys. We found both age and treatment effects on these neurons that are consistent with behavioral data. Briefly, reconstructions of pyramidal neurons in area 46 from these monkeys showed that cyclical E increased the density of small, thin spines in both young and aged monkeys. However, this effect of E was against a background of age-related loss of small, thin spines, leaving aged V-treated monkeys with a particularly low density of these highly plastic spines and vulnerable to cognitive decline. Our current interpretation is that E not only plays a critically important role in maintaining spine number, but also enables synaptic plasticity through a cyclical increase in small highly plastic spines that may be stabilized in the context of learning. Interestingly, recent studies demonstrate that chronic E is less effective at inducing spinogenesis than cyclical E. We have begun to link certain molecular attributes of excitatory synapses in area 46 to E effects and cognitive performance in these monkeys. Given the importance of synaptic estrogen receptor α (ER-α) in rat hippocampus, we focused our initial studies on synaptic ER-α in area 46. Three key findings have emerged from these studies: 1) synaptic ER-α is present in axospinous synapses in area 46; 2) it is stable across treatment and age groups (which is not the case in rat hippocampus); and 3) the abundance and distribution of synaptic ER-α is a key correlate of individual variation in cognitive performance in certain age and treatment groups. These findings have important implications for the design of hormone treatment strategies for both surgically and naturally menopausal women.
Prefrontal cortex; estrogen; aging; primate; cognition; hormone replacement therapy
Age-related memory impairment occurs in many mammalian species including humans. Moreover, women undergoing the menopausal transition often complain of problems with memory. We recently reported that rhesus monkeys display age- and menopause-related recognition memory impairment on a hippocampus-reliant test (delayed nonmatching-to-sample; DNMS). In the same monkeys, perforated synapse densities in the dentate gyrus outer molecular layer (OML) correlated with DNMS recognition accuracy, while total axospinous synapse density was similar across age and menses groups. The current study examined whether synaptic characteristics of OML axonal boutons are coupled with age- or menopause-related memory deficits. Using serial section electron microscopy, we measured the frequencies of single-synapse boutons (SSBs), multiple-synapse boutons (MSBs), and boutons with no apparent synaptic contacts (non-synaptic boutons, NSBs) in the OML. Aged females had double the percentage of NSBs as compared to young females and this measure correlated positively and inversely with DNMS acquisition (number of trials to criterion) and delay performance (average accuracy), respectively. Aged compared to young females also had a lower frequency of MSBs and a lower number of synaptic contacts per MSB, and the latter variable inversely correlated with DNMS acquisition. Although proportions of NSBs, SSBs and MSBs were similar across menses groups, compared to pre-menopausal monkeys, peri/post-menopausal monkeys had fewer MSBs contacting one or more segmented perforated synapse and the abundance of this bouton subtype positively correlated with DNMS performance. These results suggest that age- and menopause-related shifts in OML synaptic subtypes may be coupled with deficits in task acquisition and recognition memory.
delayed nonmatching-to-sample test; menopause; multiple-synapse bouton; serial sections; recognition memory
Estradiol (E) mediates increased synaptogenesis in the hippocampal CA1 stratum radiatum (sr) and enhances memory in young and some aged female rats, depending on dose and age. Young females rats express more estrogen receptor α (ERα) immunolabeling in CA1sr spine synapse complexes than aged rats and ERα regulation is E sensitive in young but not aged rats. The current study examined whether estrogen receptor β (ERβ) expression in spine synapse complexes may be altered by age or E treatment. Young (3–4 months) and aged (22–23 months) female rats were ovariectomized 7 days prior to implantation of silastic capsules containing either vehicle (cholesterol) or E (10% in cholesterol) for 2 days. ERβ immunoreactivity (ir) in CA1sr was quantitatively analyzed using post-embedding electron microscopy. ERβ-ir was more prominent postsynaptically than presynaptically and both age and E treatment affected its synaptic distribution. While age decreased the spine synaptic complex localization of ERβ-ir (i.e., within 60 nm of the pre- and post-synaptic membranes), E treatment increased synaptic ERβ in both young and aged rats. In addition, the E treatment, but not age, increased dendritic shaft labeling. This data demonstrates that like ERα the levels of ERβ-ir decrease in CA1 axospinous synapses with age, however, unlike ERα the levels of ERβ-ir increase in these synapses in both young and aged rats in response to E. This suggests that synaptic ERβ may be a more responsive target to E, particularly in aged females.
Dendrodendritic electrical signaling via gap junctions is now an accepted feature of neuronal communication in mammalian brain, whereas axodendritic and axosomatic gap junctions have rarely been described. We present ultrastructural, immunocytochemical, and dye-coupling evidence for “mixed” (electrical/chemical) synapses on both principal cells and interneurons in adult rat hippocampus. Thin-section electron microscopic images of small gap junction-like appositions were found at mossy fiber (MF) terminals on thorny excrescences of CA3 pyramidal neurons (CA3pyr), apparently forming glutamatergic mixed synapses. Lucifer Yellow injected into weakly fixed CA3pyr was detected in MF axons that contacted four injected CA3pyr, supporting gap junction-mediated coupling between those two types of principal cells. Freeze-fracture replica immunogold labeling revealed diverse sizes and morphologies of connexin-36-containing gap junctions throughout hippocampus. Of 20 immunogold-labeled gap junctions, seven were large (328–1140 connexons), three of which were consistent with electrical synapses between interneurons; but nine were at axon terminal synapses, three of which were immediately adjacent to distinctive glutamate receptor-containing postsynaptic densities, forming mixed glutamatergic synapses. Four others were adjacent to small clusters of immunogold-labeled 10-nm E-face intramembrane particles, apparently representing extrasynaptic glutamate receptor particles. Gap junctions also were on spines in stratum lucidum, stratum oriens, dentate gyrus, and hilus, on both interneurons and unidentified neurons. In addition, one putative GABAergic mixed synapse was found in thin-section images of a CA3pyr, but none were found by immunogold labeling, suggesting the rarity of GABAergic mixed synapses. Cx36-containing gap junctions throughout hippocampus suggest the possibility of reciprocal modulation of electrical and chemical signals in diverse hippocampal neurons.
CA3; dentate gyrus; interneuron; pyramidal neuron; principal cell; mossy fiber; gap junction
Hippocampal dendritic spine and synapse numbers in female rats vary across the estrous cycle and following experimental manipulation of hormone levels in adulthood. Based on behavioral studies demonstrating that learning patterns are altered following puberty, we hypothesized that dendritic spine number in rat hippocampal CA1 region would change post-pubertally. Female Sprague-Dawley rats were divided into prepubertal (postnatal day (P) 22), peripubertal (P35) and post-pubertal (P49) groups, with the progression of puberty evaluated by vaginal opening, and estrous cyclicity subsequently assessed by daily vaginal smears. Spinophilin immunoreactivity in dendritic spines was used as an index of spinogenesis in area CA1 stratum radiatum (CA1sr) of hippocampus. First, electron microscopy analyses confirmed the presence of spinophilin specifically in dendritic spines of CA1sr, supporting spinophilin as a reliable marker of hippocampal spines in young female rats. Second, stereologic analysis was performed to assess the total number of spinophilin-immunoreactive puncta (i.e. spines) and CA1sr volume in developing rats. Our results indicated that the number of spinophilin-immunoreactive spines in CA1sr was decreased 46% in the post-pubertal group compared to the two younger groups, whereas the volume of the hippocampus underwent an overall increase during this same developmental time frame. Third, to determine a potential role of estradiol in this process, an additional group of rats was ovariectomized (OVX) prepubertally at P22, then treated with estradiol or vehicle at P35, and spinophilin quantified as above in rats perfused on P49. No difference in spinophilin puncta number was found in OVX rats between the two hormone groups, suggesting that this developmental decrease is independent of peripheral estradiol. These changes in spine density coincident with puberty may be related to altered hippocampal plasticity and synaptic consolidation at this phase of maturity.
Puberty; dendritic spine; hippocampus; CA1; spinophilin; estrogen; synaptic plasticity; spinogenesis
Numerous studies have shown that neuronal plasticity in the hippocampus and neocortex is regulated by estrogen and that aromatase, the key enzyme for estrogen biosynthesis, is present in cerebral cortex. Although the expression pattern of aromatase mRNA has been described in the monkey brain, its precise cellular distribution has not been determined. In addition, the degree to which neuronal aromatase is affected by gonadal estrogen has not been investigated. In this study, we examined the immunohistochemical distribution of aromatase in young ovariectomized female rhesus monkeys with or without long-term cyclic estradiol treatment. Both experimental groups showed that aromatase is localized in a large population of CA1-3 pyramidal cells, in granule cells of the dentate gyrus and in some interneurons in which it was co-expressed with the calcium binding proteins calbindin, calretinin, and parvalbumin. Moreover, numerous pyramidal cells were immunoreactive for aromatase in the neocortex, whereas only small subpopulations of neocortical interneurons were immunoreactive for aromatase. The widespread expression of the protein in a large neuronal population suggests that local intraneuroral estrogen synthesis may contribute to estrogen-induced synaptic plasticity in monkey hippocampus and neocortex of female rhesus monkeys. In addition, the apparent absence of obvious differences in aromatase distribution between the two experimental groups suggests that these localization patterns are not dependent on plasma estradiol levels.
aromatase; estrogen; hippocampus; interneuron; neocortex; pyramidal cells
While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.
adult; antigen retrieval; blood vessels; brain; collagen IV; immunohistochemistry; mouse; pepsin
17β-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor α (ER-α) that potentially regulate spine formation, such as LIM-Kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-α, phospho-LIMK (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (pLIMK-IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (p<0.02) in young animals, and decreased (43%) with age (p<0.002) regardless of treatment. The pattern of distribution of pLIMK-IR within dendritic spines and synapses was unaffected by age or E treatment, with the exception of an E-induced increase in the non-synaptic core of spines in young females. These data suggest that age-related synaptic alterations similar to those seen with ER-α occur with signaling molecules such as pLIMK, and support the hypothesis that age-related failure of E treatment to increase synapse number in CA1 may be due to changes in the molecular profile of axospinous synapses with respect to signaling pathways linked to formation of additional spines and synapses in response to E.
sex steroids; electron microscopy; immunogold; signal transduction; plasticity; synapse
Synapses onto dendritic spines in the lateral amygdala formed by afferents from the auditory thalamus represent a site of plasticity in Pavlovian fear conditioning. Previous work has demonstrated that thalamic afferents synapse onto LA spines expressing glutamate receptor (GluR) subunits, but the GluR subunit distribution at the synapse and within the cytoplasm has not been characterized. Therefore, we performed a quantitative analysis for ∝-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptor subunits GluR2 and GluR3 and N-methyl-D-aspartate (NMDA) receptor subunits NR1 and NR2B by combining anterograde labeling of thalamo-amygdala afferents with postembedding immunoelectron microscopy for the GluRs in adult rats. A high percentage of thalamo-amygdala spines was immunoreactive for GluR2 (80%), GluR3 (83%), and NR1 (83%), while a smaller proportion of spines expressed NR2B (59%). To compare across the various subunits, the cytoplasmic to synaptic ratios of GluRs were measured within thalamo-amygdala spines. Analyses revealed that the cytoplasmic pool of GluR2 receptors was twice as large compared to the GluR3, NR1 and NR2B subunits. Our data also show that in adult brain, the NR2B subunit is expressed in the majority of in thalamo-amygdala spines and that within these spines, the various GluRs are differentially distributed between synaptic and non-synaptic sites. The prevalence of the NR2B subunit in thalamo-amygdala spines provides morphological evidence supporting its role in the fear conditioning circuit while the differential distribution of the GluR subtypes may reflect distinct roles for their involvement in this circuitry and synaptic plasticity.
GluR2; GluR3; excitatory amino acids; immunogold; NR1; NR2B; postembedding; immunohistochemistry; tracing; electron microscopy
It is believed that gene/environment interaction (GEI) plays a pivotal role in the development of motor skills, which are acquired via practicing or motor training. However, the underlying molecular/neuronal mechanisms are still unclear. Here, we reported that the expression of NR2B, a subunit of NMDA receptors, in cerebellar granule cells specifically enhanced the effect of voluntary motor training on motor learning in the mouse. Moreover, this effect was characterized as motor learning-specific and developmental stage-dependent, because neither emotional/spatial memory was affected nor was the enhanced motor learning observed when the motor training was conducted starting at the age of 3 months old in these transgenic mice. These results indicate that changes in the expression of gene(s) that are involved in regulating synaptic plasticity in cerebellar granule cells may constitute a molecular basis for the cerebellum to be involved in the GEI by facilitating motor skill learning.
In rat hippocampus, estrogen receptor-alpha (ER-α) can initiate non-genomic signaling mechanisms that modulate synaptic plasticity in response to either circulating or locally synthesized estradiol (E). Here we report quantitative electron microscopic data demonstrating that ER-α is present within excitatory synapses in dorsolateral prefrontal cortex (dlPFC) of young and aged ovariectomized female rhesus monkeys with and without E treatment. There were no treatment or age effects on the percentage of excitatory synapses containing ER-α, nor were there any group differences in distribution of ER-α within the synapse. However, the mean size of synapses containing ER-α was larger than unlabeled excitatory synapses. All monkeys were tested on delayed response (DR), a cognitive test of working memory that requires dlPFC. In young ovariectomized monkeys without E treatment, presynaptic ER-α correlated with DR accuracy across memory delays. In aged monkeys that received E treatment, ER-α within the postsynaptic density (30–60 nm from the synaptic membrane) positively correlated with DR performance. Thus, while the lack of group effects suggests that ER-α is primarily in synapses that are stable across age and treatment, synaptic abundance of ER-α is correlated with individual performance in two key age/treatment groups. These data have important implications for individual differences in the cognitive outcome among menopausal women and promote a focus on cortical estrogen receptors for therapeutic efficacy with respect to cognition.
estradiol; menopause; aging; cognition; dendritic spines; electron microscopy